Cutaneous melanoma (CM) is one of the malignant tumors with a relative high lethality. Necroptosis is a novel programmed cell death that participates in anti-tumor immunity and tumor prognosis. Necroptosis has been found to play an important role in tumors like CM. However, the necroptosis-associated lncRNAs’ potential prognostic value in CM has not been identified.The RNA sequencing data collected from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression Project (GTEx) was utilized to identify differentially expressed genes in CM. By using the univariate Cox regression analysis and machine learning LASSO algorithm, a prognostic risk model had been built depending on 5 necroptosis-associated lncRNAs and was verified by internal validation. The performance of this prognostic model was assessed by the receiver operating characteristic curves. A nomogram was constructed and verified by calibration. Furthermore, we also performed sub-group K-M analysis to explore the 5 lncRNAs’ expression in different clinical stages. Function enrichment had been analyzed by GSEA and ssGSEA. In addition, qRT-PCR was performed to verify the five lncRNAs’ expression level in CM cell line (A2058 and A375) and normal keratinocyte cell line (HaCaT).We constructed a prognostic model based on five necroptosis-associated lncRNAs  and divided patients into high-risk group and low-risk group depending on risk scores. A predictive nomogram had been built to be a prognostic indicator to clinical factors. Functional enrichment analysis showed that immune functions had more relationship and immune checkpoints were more activated in low-risk group than that in high-risk group. Thus, the low-risk group would have a more sensitive response to immunotherapy.This risk score signature could be used to divide CM patients into low- and high-risk groups, and facilitate treatment strategy decision making that immunotherapy is more suitable for those in low-risk group, providing a new sight for CM prognostic evaluation. CM is the result of a genetic mutation caused by ultraviolet ray radiation . Though Necroptosis is a type of controlled cell death that mimics both necrosis and apoptosis. Plasma membrane permeabilization occurs rapidly during necroptosis. In this process, the cell content is released and then subsequently exposed to a variety of cytokines, chemokines, and damage-associated molecular patterns. The immunogenic nature of necroptotic cancer cells and their capacity to effectively trigger anti-tumor immunity are both attributed to necroptosis, which is becoming increasingly recognized as being crucial in cancer , 6. For Long non-coding RNAs (lncRNAs) are those having transcripts that are 200 nucleotides or longer. Multiple studies have shown that lncRNAs are involved in cancer. lncRNA-Gm31932 uses the miR-344d-3-5p/Prc1 axis, for example, to induce cell cycle arrest and differentiation in melanoma . LncRNA To create a unique predictive signature, this work analyzed the relationship between necroptosis-associated lncRNAs and clinicopathological features, and immune infiltration of individuals with CM. Internal verification and gene enrichment analysis (GSEA) were used to assess the robustness of the model as well as the potential mechanisms, respectively.22.1The Cancer Genome Atlas  was used to acquire RNA transcriptome datasets and associated clinical information of CM, and a synthetic data matrix concerning healthy skin data was obtained from Genotype-Tissue Expression Project . The data were merged and processed using the R “Limma” tool. Furthermore, the lncRNA expression values and survival rates for 471 CM patients were determined.2.2https://www.genecards.org/) and other published studies  and necroptosis-associated lncRNAs in CM, the “survival” R package was used to perform a univariate Cox regression (uni-Cox) analysis at a significance level of p < 0.001. Then, the R package “caret” was employed to randomly classify the CM samples into the training and testing cohorts.The screening criteria for identifying the necroptosis-associated lncRNAs in CM samples with expression values were the correlation coefficients |R| > 0.3 and To identify the most important necroptosis-associated lncRNA with CM patients, we performed LASSO-penalized Cox regression analysis by the “glmnet” R package. The selection of variables in the Cox model was performed using the lasso method, and a risk signature was generated using the “survminer” package in R. The following calculations were utilized to determine the risk score: Risk score = sum (each lncRNA’s expression × corresponding coefficient). Low-risk (LR) and high-risk (HR) groups of CM patients were defined using the median risk score in both the training and testing datasets. The clinical information for the entire set was shown in The relationship between candidate lncRNA and mRNA was visualized by the Cytoscape diagram. Furthermore, “gg alluvial” in the R package was employed to visualize the distribution of the 5 candidate genes in LR and HR groups. The Kaplan-Meier (K-M) survival analysis and the correlation between risk score and survival time were performed using the “survival” and “survminer” packages in R. 1-year, 3-year, and 5-year ROC analyses were conducted using the “timeROC” R-package.The “survival” R-package was used to create a prediction model, which included univariate and multivariate independent prognostic studies to determine the correlation between clinical features, risk score, and patient OS. A heatmap was made to illustrate the distribution of clinical features and potential lncRNAs in LR and HR groups. ROC analysis on risk scores and clinical features was done with the “survival ROC” package.2.4Using the risk score, age, and T, N stage, the R-package “rms” was used to create a nomogram for 1-year, 3-year, and 5-year OS. Using a calibration chart and ROC curves, we analyzed the nomogram’s prognostic accuracy.2.5p<0.05). Here, the GSVA software and ssGSEA were used to determine the infiltration scores of 16 immunological cells and the 13 immune-related pathway activities.The predominant route genes were analyzed using GSEA. GSEA 4.1.0 was employed to conduct the analysis. The cutoffs for statistical significance were minimal  and human melanoma cell lines (A2058 and A375) were purchased from American Type Culture Collection (ATCC). HaCaT and A375 cell lines were cultured in DMEM (Dulbecco’s Modified Eagle Medium)  and A2058 cell line was cultured in DMEM/F-12  and both were added 10% fetal bovine serum  at 37°C in an incubator with 5% CO2.2.7® III 1st Strand cDNA Synthesis SuperMix  according to standard protocol. The cDNA was used as a template and lncRNA expression was quantified using SYBR Green Master Mix . The primer pairs were synthesized by Tsingke Biotechnology , and the primer pairs are listed in −ΔΔCt method was used to evaluate relative expression levels.Total RNA of the three cell lines was extracted with TRIzol Reagent . cDNA was synthesized with Hifair2.8p < 0.05 level.R 4.0.2 version and Prism 5 were used to conduct all statistical analyses. Analysis of continuous data was conducted using the Wilcoxon test, while analysis of categorical data was conducted using the Chi-square or Fisher tests. The log-rank test and the K-M technique were used to compare the OS rates of patients in the HR and LR groups. The collected findings were also considered significant at the 33.1p<0.001) were identified by comparing the expression of 201 necroptosis-associated genes to 16,977 DEGs (|Log 2 FC|>1 and p<0.05) between normal and CM samples (3.2p<0.001) (We identified 880 necroptosis-associated lncRNAs Table S3p<0.001). ROC study indicated that the risk signature had reasonable predictive accuracy at 1-year (ROC = 0.758), 2-year (ROC = 0.701), and 3-year (ROC = 0.727) (3.3p<0.001) in uni-Cox regression, respectively, 1.385 and 1.260-1.523 (p<0.001) in multi-Cox regression (p<0.001), T stage , and N stage  (Univariate and multivariate Cox regression (multi-COX) analyses show that the newly identified risk signature is an independent prognostic factor for CM patients. The hazard ratio (HR) of the risk score and 95% confidence interval (CI) were 1.354 and 1.240-1.478 (3.4p<0.05 in multi-Cox) to predict the 1-, 3-, and 5-year OS incidences of CM patients , the OS of patients in the HR groups was much lower than that of patients in the LR groups, demonstrating that the predictive signature can reliably predict the prognosis of CM patients (3.6p<0.0001)  and testing (N=226) cohorts using the same algorithm and regression coefficient (β). As expected, the K-M survival analysis confirmed what was observed in the entire dataset: CM patients in the LR group had longer OS than those in the HR group in both the training and testing cohorts , natural killer cells (NK cells), macrophages, and almost all immunological activity were more strongly activated in the LR group -anchor biosynthesis, cell TCA cycle, and aminoacyl-tRNA biosynthesis. While the samples from the group in the LR group are richer in antigen processing and presentation, cell adhesion, and chemokine signaling  and a normal keratinocyte cell line (HaCaT) to compare the lncRNAs’ expression. The expression of LINC00665 and AC018553.1 in both melanoma cell lines are higher than that in HaCaT . And these lncRNAs are experimentally verified by qRT-PCR. In these groups, the patients in the HR group had a shorter OS than those in the LR group. Subgroups based on pathological types, grades, M, and N were all shown to be of comparable importance in the prognostic analysis. The multi-Cox regression model identified the necroptosis-associated lncRNA signature as a significant independent risk factor for CM prognosis. A nomogram map was constructed to predict the survival of CM patients at 1, 3, and 5 years. The risk scores are the representation of the clinical outcomes. There was a poor prognosis for patients who scored highly on the necroptosis-associated lncRNAs signature. Also, compared with previously established signature, our signature has a better performance in AUC value . ssGSEA via the miR-339-3p/TUBB axis (Among these five lncRNAs, LINC01871 and AC107464.3 are protective factors, while AC245041.1, AC018553.1, and LINC01871 are risk factors. And these findings were experimentally verified by qRT-PCR. These lncRNAs are widely studied in different types of cancer. For example, AC245041.1 has been certificated to participate in angiogenesis, cell adhesion, wound healing, and extracellular matrix organization processes in stomach adenocarcinoma . By reguUBB axis . AC01855UBB axis , 30. MeaUBB axis . LINC018UBB axis , autophaUBB axis . As was + T cells, DCs, NK cells, and macrophages. Based on single-cell studies, Sukumar et al. shows melanoma reactivity is linked to a high level of dysfunctionality in CD8+ T cells. A rise in CD8+ T cell memory, on the other hand, is associated with anti-melanoma effects (+ T cells release perforin and granules to induce melanoma cell apoptosis (+ DCs are proven to be critical tumor-draining antigen-presenting cells driving CD8+ T cells to elicit strong T cell response in melanoma (+ tumor-infiltrating lymphocyte expansion and also effector-memory (The efficiency of immunological functions is greatly influenced by immune cells, 36. Our effects . CD8+ T poptosis . In prevmelanoma , 40. NK melanoma , 42. Mormelanoma . Differemelanoma . It has r-memory , 46. TheThe immunological checkpoint typically has a detrimental effect on immune system control, which is essential for preserving self-tolerance . HoweverAccording to earlier research, the immune system changed during melanomagenesis and reduced anti-tumor immunity . This stHowever, the model we developed still had certain weaknesses despite our attempts to address them using several different approaches. There were problems with the research that was inevitable given that it was conducted in hindsight. Additionally, further research is needed that integrates biochemical tests with clinical prognostic information to properly identify how these lncRNAs impact the prognosis of CM patients through necroptosis.In conclusion, a signature of five necroptosis-associated lncRNAs was created in this investigation to predict the prognosis of CM. Our results also indicated that NALncSig is a separate risk factor for CM. We hope to provide a new reference for the current prognostic assessment of CM and to shed new light on treatment strategies.The original contributions presented in the study are included in the article/Conception and design: ZWC and ZL; acquisition, analysis, or interpretation of data: GC, BYL, and YNG; drafting the work: ZWC, BYS, and YHZ; revising the work: JGM and BQS. All authors contributed to the article and approved the submitted version.


Background: This study aims to determine the factors affecting HBV DNA suppression in chronic hepatitis B patients with tenofovir disoproxil fumarate (TDF).Methods: A case-control was carried out from October 2021 to August 2022 on 182 chronic hepatitis B patients who had TDF therapy regularly for 24 weeks at H. Adam Malik and USU Hospitals in Medan, Indonesia. The history of the samples was obtained, followed by physical examination, and blood collection. CTLA-4 polymorphism examination was carried out using real-time PCR, while the serum CTLA-4 levels were assessed with ELISA.Results: The CTLA-4 -1661G>A polymorphism, genotype GG+AG, increased 1.52 times risk of not achieving HBV DNA suppression to TDF compared to genotype AA (p=0.041). High CTLA-4 levels increased 2.28 times risk, high HBV DNA levels increased 2.09 times risk, low ALT levels increased 1.95 times risk of not achieving HBV DNA suppression . There was no relationship between gender, age, ethnicity, obesity, baseline AST, HBeAg, genotype, liver fibrosis and HBV DNA suppression after 24 weeks of treatment (p>0.05).Conclusions: The levels of CTLA-4, HBV DNA, ALT, and CTLA-4 -1661G>A polymorphism have a potential relationship with the suppression of HBV DNA in chronic hepatitis B patients with TDF. Hepatitis B (HBV) is a health problem affecting several countries in the world, including Indonesia. A national survey in 2013 showed the proportion of positive Hepatitis B surface antigen (HBsAg) was 7.1% . The ideCTLA-4 levels in the body are influenced by allele variations in the CTLA-4 gene, which is located on chromosome 2q33.3 and consists of 4 exons and 3 introns . Chen's These studies have focused on the association of CTLA-4 polymorphism with the persistence and progression of chronic hepatitis B infection, but to our knowledge, no studies have explored its relationship with HBV DNA suppression. Therefore, this study aims to determine the association of the CTLA-4-1661G>A gene polymorphism and other factors, such as age, gender, ethnicity, obesity, baseline AST, baseline ALT, baseline HBV DNA, HBeAg, genotype, degree of fibrosis, and CTLA-4 levels with HBV DNA suppression in chronic hepatitis B patients treated with TDF.A case-control study was carried out from October 2021 to August 2022 on 182 chronic hepatitis B patients who had already done 24 weeks therapy with TDF at H. Adam Malik and USU Hospitals in Medan, Indonesia. Medication adherence was observed by self-reporting diary. The case group was patients who did not achieve HBV DNA suppression after 24 weeks of TDF treatment. The control group was patients who achieved HBV DNA suppression after 24 weeks of TDF treatment. The research samples were taken using consecutive sampling technique. All subjects who came and met the study criteria were included consecutively in the case or control group until the number of samples was fulfilled.th, 2021 . Written informed consent was obtained from participants before enrollment in this study  = 0.57, p2 (Proportion of patients with the GG+GA genotypes of CTLA-4-1661G>A polymorphism who achieve HBV DNA suppression) = 0.23 . The HBeAg and anti-HBe antibody levels were examined using a commercial enzyme-linked immunosorbent assay (ELISA) kit . The HBV DNA viral load was assessed with the COBAS TaqMan HBV Test, version 2.0 , with a measurement ratio of 2.1 – 9 log copies/mL. We used the kits according the manufacturer’s instructions.DNA extraction was carried out with Genomic DNA Mini Kit , Cat GB100, Lot. FG02009) using whole blood approximately 5 ml. All the procedure for CTLA-5-1661 G>A were performed in Prodia Laboratorium, Medam, Indonesia. All the procedure under the working procedure according to the kit.The Primer & Probe used in this study were TaqMan SNP Genotyping Assay CTLA-4 -1661 G>A  cat:44033111, Lot. 00905960. Genotyping of the CTLA-4-1661G>A gene was performed with a forward primer: 5′-CTAAGAGCATCCGCTTGCACCT-3′ and a reverse primer: 5′ TTGGTGTGATGCACAGAAGCCTTTT-3′.TM RT-PCR (Biorad) with the following standard protocol: 20 seconds of enzyme activation at 95°C, followed by 40 amplification cycles consisting of 15 seconds of denaturation at 95°C, and 1 minute of annealing/extension at 60 C.Amplification was performed with a C1000 thermal cycler CFX96 real-time system  using the following standard protocol. The reaction mixture for RT-PCR used the TaqMan GTXpress master mix (2x) reagent with a total volume of 25 ul TaqMan GTXpress Master Mix (2x), added 1.25 ul 20x working stock SNP genotyping assay, 6.25 ul ddH20, and 5 ul DNA template with a concentration of 1-10ng/well. Amplification was performed using a CFX 96 touch(R) ELISA with catalog number HSCT40, Human CTLA-4 Immunoassay R&D System Inc., Minneapolis, USA). All the materials we need, namely human CTLA-4 capture antibody (lyophilized), human CTLA-4 detector antibody 10x, human CTLA-4 lyophilized recombinant protein, antibody diluent 4BI, cell extraction buffer PTR 5X, cell extraction buffer enhancer solution 50X, stop solution, TMB development solution, wash buffer PT 10X, and sample diluent NS. All the items store in 4 C.The measurement of serum CTLA-4 levels was performed with the ELISA method , open the seal plate, put it back in the foil pouch, and store it at 4°C. Then add 50 μL of sample or standard to each well. After that, add 50 μL Antibody Cocktail to each well.The plate was closed with a seal, then incubated for 1 hour at room temperature on a shaker at 400 rpm. Afterward, wash each well with 3 x 350 μL 1X Wash Buffer. In the last washing, turn the plate over and dry, then add 100 μL TMB Development Solution to each well and incubate for 10 minutes in the dark on a shaker at 400 rpm. Adding the Stop Solution will change the color from blue to yellow and increase the signal intensity by about 3X. To avoid signal saturation, proceed to the next step before the high concentration of standard reaches a blue color OD 600 equals 1.0. Given the variability in laboratory environmental conditions, the optimal incubation time can vary between 5 and 20 minutes.In the final stage, add 100 μL Stop Solution to each well. It alternates the plate so that it is homogeneous. Then read at a wavelength of 450 nm.Data analysis was carried out using the chi-square test and binomial logistic regression. Hardy Weinberg equilibrium was tested by chi square analysis. The significance of the statistical test results was determined based on the p-value <0.05 using SPSS-22 Statistics for Windows . Cut off used in age classification, AST level, ALT levels, HBV DNA based on the mean of the study sample was 44 years, 114 IU/L, 120 IU/L, and 6.31 log 10 IU/mL. Cut off for CTLA-4 classification based on the results of the ROC curve analysis.There was a significant relationship between low baseline ALT, high baseline HBV DNA, and high CTLA-4 levels with not achieving HBV DNA suppression (p<0.05) ,2022b. CTLA-4 levels were significantly higher in chronic hepatitis B patients without HBV DNA suppression compared to others (p=0.016), as shown inThe accuracy of CTLA-4 in predicting HBV DNA suppression in chronic hepatitis B patients with TDF is presented inCTLA-4 levels of 52.2 pg/mL were able to predict the suppression of HBV DNA by TDF with a sensitivity and specificity of 60.4% and 62.6%, respectively, as shown inThe genotype frequency of the -1661G>>A of CTLA-4 polymorphism in the case group (p= 0.987) and control group (p= 0.879) in Hardy Weinberg equilibrium (p>0.05) as shown inThere was a significant relationship between the CTLA-4-1661G>A polymorphism and HBV DNA suppression. The GG+AG genotype increased 1.42 times risk of not achieving HBV DNA suppression compared to the AA genotype (p=0.031). While there was no relationship between the GG genotype and HBV DNA suppression compared to the AG+AA genotype. Furthermore, patients with the G allele were 1.27 times more at risk compared to others with the A allele (p=0.025), as shown inThe multivariate analysis showed that the levels of CTLA-4, HBV DNA, ALT, and CTLA-4 -1661G>A polymorphisms are associated with the suppression of HBV DNA. Furthermore, high CTLA-4 levels significantly increased the risk of not achieving suppression by 2.28 times (p=0.009). High HBV DNA significantly increased the risk by 2.09 times (p=0.026), while patients with low baseline ALT are 1.95 times more at risk (p=0.036). People with polymorphism CTLA-4 -1661G>A genotype GG+AG are 1.52 times more at risk of not achieving HBV DNA suppression (p=0.041), as shown inBased on the B values obtained from the calculation above, the equation model formed was: Ln P/1-P = -7.815 + 2.055 high CTLA-4 + 1.722 high HBV DNA + 1.599 low ALT + 1.204 CTLA-4 polymorphism - 1661G>A. The derivative formula of the equation can also be used, namely Probability = exp (-7.815 + 2.055 high CTLA-4 + 1.722 high HBV DNA + 1.599 low ALT + 1.204 CTLA-4 polymorphism -1661G>A) /1 + exp (-7.815 + 2.055 high CTLA-4 + 1.722 high HBV DNA + 1.599 low ALT + 1.204 CTLA-4 polymorphism -1661G>A).There were significant differences in the serum CTLA-4 levels between the genotypes of CTLA-4-1661G>A polymorphism. Furthermore, CTLA-4 levels were significantly higher in hepatitis B patients with genotypes GG and AG compared to others with AA (p<0.001). It was also significantly higher in people with the G allele compared to the A allele (p<0.001), as shown inChronic hepatitis B is a health problem affecting several countries in the world, including Indonesia and it has high morbidity and mortality due to its complications. Furthermore, the importance of management in patients with the disease is to achieve treatment endpoints and to prevent its complications. Tenofovir disoproxil fumarate (TDF) is one of the NA classes of drugs used as the first line of treatment . It alsoMen are more prone to chronic hepatitis B infection due to immune, hormonal, and risk factors . KosinskKennedy stated that the age group with the worse T-cell response to HBV was children . This is in line with previous studies that ethnicity/race has no relationship with virological response to NA or Peg-IFN .Animal studies showed that obese mice have lower levels of antibodies and T cells compared to non-obese mice , while patients with the G allele of CTLA-4 -1661G>A have a 1.27-fold increased risk compared to others with the A allele (p=0.025). Based on the multivariate analysis, people with genotype GG+AG are 1.52 times more at risk of not achieving HBV DNA suppression (p=0.041). The effect of the CTLA-4-1661G>A gene polymorphism on virological response to TDF is caused by an increase in CTLA-4 level. Furthermore, CTLA-4 molecules through their interactions with costimulatory molecules B7 can cause negative regulation of T cells. Their increased expression can also increase spontaneous T cell apoptosis. CTLA-4 can influence infection progression and antiviral immune response . TreatmeThis study demonstrated a dose-dependent effect of the G allele in increasing serum CTLA-4 level as well as the risk of not achieving a virological response to TDF. Moreover, CTLA-4 production and risk of non-responders to TDF were high in patients with the GG genotype, intermediate in AG, and low in AA, while the A allele was protective.This study had limitations, including not analyzing quantitative HBeAg, quantitative HBsAg, and polymorphisms of other genes that can affect the virological response to TDF. This study also only analyzed 1 location of the CTLA-4 gene, namely -1661G>A.The CTLA-4 -1661G>A polymorphism can be used as a predictor of chronic hepatitis B treatment response so that appropriate management and ongoing monitoring can be done to achieve treatment targets and prevent the progression of chronic hepatitis B. Further research is needed to determine the role of anti-CTLA-4 in increasing the virological response to TDF.The results showed the factors affecting HBV DNA suppression in chronic hepatitis B patients with tenofovir disoproxil fumarate (TDF) were the levels of CTLA-4, HBV DNA, ALT, and CTLA-4-1661G>A polymorphism. There was no relationship between gender, age, ethnicity, obesity, baseline AST, HBeAg, genotype, liver fibrosis and HBV DNA suppression after 24 weeks of treatment (p>0.05). This revised article accommodates my suggestions.  I have no further comment.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Hepatology and gastroenterologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I have read the revised article and approve to the revised article without any further revision.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Darmadi et al have done a very good and useful study to find out factors affecting HBV DNA suppression in chronic hepatitis B treated with Tenofovir Disoproxil Fumarate (TDF). TDF is one of the first-line drugs used for treatment of chronic hepatitis B. Previous studies have shown longterm nucleotide analog therapy (eg: TDF) can increase peripheral T cell levels to enhance antiviral response. Their activation is strongly determined by genetic factors, such as CTLA-4. It's really interesting that Darmadi et al focused on the association of CTLA-4 polymorphism in the persistence and progression of chronic hepatitis B infection, with HBV DNA suppression. Although this study has so many good points, there might be some suggestions and comments to improve this research article. First of all, there is a similarity in the abstract at the results and conclusion section. Preferably, the results are presented with numerical data. Then, in the methods, it is stated that "the case group was patients who did not achieve virological response to TDF treatment, the control group was patients who achieved a virological response to TDF treatment." The author need to give definition of virological response to NUCs (eg. Tenofovir). Inclusion criteria was also did not mention in the Methods section. In the procedure section, CTLA-4 level was measured, but the author did not state when the measurement was done. The author should state whether the measurement was done before, after, or before and after the treatment started. In the data analysis, it is important to explain the cut off and how to define each of the categorical variables, eg. the age is divided into old and young, the author need to state the criteria of old and young. The AST and ALT baseline is better categorized as normal and increased instead of low and high. In the discussion section, the author has written down the basic theory and supporting studies that are advanced and complete. However, it is better to use the latest reference to optimize and support this study as there are several references dates up to 24 years ago. In the discussion it should be stated how this research may change the daily clinical practice of clinicians. Does CTLA-4 levels or CTLA-4 polymorphism need to be checked for every chronic hepatitis B patients naive-treatment to achieve ideal or satisfactory end point or just to measure HBV DNA suppression.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Hepatitis B, Liver Cirrhosis, Hepatocellular CarcinomaI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The results of the study in the abstract should be mentioned clearly with the data, not the same as the conclusions.Some of the reference literature is older than 10 years. The aim of the study, the results, and the conclusion should be in line.Please use the term relation, correlation, and association properlyDescribe the study design clearly. This is a case-control study, how to find and determine the case and control to reduce selection bias. how to find 91 cases and 91 control out of 276 samples ( after doing exclusion ). And not clear when you did the test of CTLA-4, at the start of treatment or at the end of treatment?Please state clearly, how to determine categorical variables from the numerical variable. ex: High vs Low in AST, ALT, DNA etc. or high vs normal?Table 5 should be presented clearlylow baseline ALT,high baseline HBV DNA,high CTLA-4 levels, and HBV DNA suppression (p<0.05) ." " low baseline ALT are 1.37 times more at risk ofnot achieving HBV DNA suppression (p=0.037)," Please make a clear statement in the text to avoid misinterpretations. ex. in the results: "There was a significant relationship betweenI think this paper is excellent and is an important addition to the literature. The new idea in determining the response to hepatitis B treatment is important. but I have some suggestions for this paper:Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Hepatology and gastroenterologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.


Toxocara canis (T. canis). The efficacy of the treatment was assessed in the acute and chronic phases of the infection by larval recovery and histopathological, immunohistochemical, and biochemical studies. The results showed that combined therapy significantly reduced larval counts in the liver, brain, and muscles and ameliorated hepatic and brain pathology. It reduced oxidative stress and TGF-β mRNA expression and increased FGF21 levels in the liver. It decreased TNF-α levels and MMP-9 expression in the brain. In addition, it increased serum levels of IL-12 and IFN-γ and decreased serum levels of IL-4 and IL-10. In the acute and chronic phases of the infection, the combined treatment was more effective than ABZ alone. In conclusion, this study highlights the potential role of MTF as an adjuvant in the treatment of experimental T. canis infection when administered with ABZ.There are currently insufficient anthelmintic medications available for the treatment of toxocariasis. For instance, Albendazole (ABZ) is the preferred medication, but its effectiveness against tissue-dwelling parasites is limited. In addition, Metformin (MTF) is a widely used oral antidiabetic medication that is considered to be safe for treatment. This study aimed to investigate any potential effects of MTF, alone or in combination with ABZ, on mice infections caused by  T. canis larvae :  suspension  which contains 100 mg/5 ml was used. Each mouse received 100 mg/kg daily via oral gavage for five consecutive days  were used. The mice were kept in typical cages at a temperature of 25 ± 2 °C, were fed a standard diet, and had free access to water. All mice were exposed to a 7-day acclimation period prior to the experiment.Negative control group includes Group (I) (10 mice) uninfected control group.Acute toxocariasis groups (A) (5 mice each) include Group (A/II) acute infected untreated control group (acute positive control), Group (A/III) treated with ABZ alone starting on the 8th day PI. (acute ABZ), Group (A/IV) treated with MTF alone starting on the 8th day PI (acute MTF), and Group (A/V) treated with ABZ and MTF starting on the 8th day PI. MTF was given in parallel with ABZ and then continued for the following ten days (acute combined).Chronic toxocariasis groups (C) (5 mice each) include Group (C/II) chronic infected untreated control group (chronic positive control), Group (C/III) treated with ABZ alone starting on the 71st day PI (chronic ABZ), and Group (C/IV) treated with MTF alone starting on the 71st day PI (chronic MTF), and Group (C/V) treated with ABZ and MTF starting on the 71st day PI. MTF was given in parallel with ABZ and then continued for the following 10 days (chronic combined).The following groups of mice were used:All mice of groups  and 5 mice from group (I) were sacrificed on the 22nd day PI, and all mice of groups  and 5 mice of group (I) were sacrificed on the 85th day PI. In order to do a parasitological investigation on all infected mice, larvae were counted in half of the liver, half of the brain, and muscles. For histopathological and immunohistochemical tests, identical brain and liver samples from each mouse were taken and stored in 10% formalin. Samples of each mouse’s brain, liver, and serum were collected and stored at −80 °C for biochemical analysis.According to Liao et al. , larval Percentage of reduction = 100 × (NC − NT)/NCNC is the mean larval count in the corresponding control group/mouse; NT is the mean larval count in the treated group/mouse.https://eliceirilab.org/software/imagej/). The mean diameter of granulomas per hepatic section was calculated  in 3 different fields per mouse to calculate the average. The largest diameter was measured using the ImageJ software  expression was assessed by immunohistochemistry in accordance with the manufacturer’s instructions using the ultra-vision detection Kit  and rabbit polyclonal anti-MMP-9 .A brown granular cytoplasmic stain was identified as MMP-9 positive immunostaining. The number of stained cells and the degree of staining were taken into account when calculating the score. Scores for staining intensity ranged from 0 to 3. 0 signified no staining, 1 slight staining, 2 moderate staining, and 3 high staining. On a scale of 1 to 4, the frequency of stained cells was rated as follows: 1 = (0–25%), 2 = (26–50%), 3 = (51–75%), and 4 = (75–100%). The intensity score and frequency score were multiplied to produce the final score. According to the final score, the staining was divided into five categories: score 0, absent expression; score +1 (1–2); score +2 (3–4); score +3 (6–8); and score +4 (9–12)  (CAT# NO 25 33), Malondialdehyde (MDA) (CAT# MD 25 29), superoxide dismutase (SOD) (CAT# SD 25 21), and catalase (CAT# CA 25 17) levels were determined in liver tissue homogenate using kits provided by Bio-diagnostic . Using ELISA kits, the levels of liver homogenate fibroblast growth factor 21 (FGF21) , brain homogenate tumor necrosis factor-alpha (TNF-α) , serum interleukin-12 (IL-12) , serum interferon-gamma (IFN-γ) , serum interleukin-4 (IL-4) , and serum interleukin-10 (IL-10)  were determined. All ELISA techniques were read using a microplate reader  at 450 nm with a correction wavelength set at 570 nm per the manufacturer’s recommendations.RNA was extracted from frozen liver homogenate using a Qiagen RNeasy Total RNA isolation kit  and following the manufacturer’s instructions. The first strand of DNA was synthesized using the Super-Script III First-Strand Synthesis System for a real-time PCR kit  and following the manufacturer’s instructions. PCR reactions were conducted using the Power SYBR Green PCR Master Mix  according to the manufacturer’s instructions. The TGF-mRNA transcript amount was measured utilizing the housekeeping gene TGF-β as an internal control. These sequence-specific primers were developed as follows: mice TGF-β (Gene Bank accession NM_011577.2): up-stream: 5′-CTCTCTGCTCCTCCCTGTTCTA-3′, down-stream: 5′-ATAGATGGCGTTGTTGCGGT-3′ with amplicon size 202 bp, mice β-actin (Gene Bank accession NM_007393.5): up-stream: 5′-TTACAGGAAGTCCCTCACCC-3′, and down-stream: 5′-ACACAGAAGCAATGCTGTCAC-3′ with amplicon size 110 bp. Relative gene expression was calculated automatically by using the comparative threshold (Ct) method for the values of the target and the reference genes using the 2-ΔΔCT formula , software for Windows, version 18.0, was used to perform the statistical analyses.The data were displayed as means ± standard deviation. One-way ANOVA was used to compare more than two groups, and the post hoc test was used to find the likelihood of significant differences between the dual means of the groups. The chi-square test was used to compare qualitative data between the two groups. Values were considered significant when At both time points PI, the mean number of larvae detected in the brain and muscles of the treated groups was significantly lower than that of the positive control group. The combined group had the best results, with reduction percentages in the brain (62.0% and 58.0%) and muscles (79.7% and 73.2%) during the acute and chronic phases of infection, respectively. The combined treatment was significantly more effective than each drug administered separately. In the acute phase of infection, either ABZ or MTF alone significantly decreased liver larval counts. Nevertheless, neither drug significantly reduced the number of hepatic larvae in the chronic phase of the infection. The combined treatment was successful in reducing the number of larvae in the liver, with reduction percentages of 82.6% and 84.6% in the acute and chronic stages, respectively Table .Table 1In comparison to the positive control group, the mean number of granulomas per hepatic section decreased significantly in all ABZ-treated and MTF-treated groups. However, the difference between acute ABZ (G A/III) and acute MTF (G A/IV) groups was statistically insignificant. Furthermore, the combined groups significantly decreased when compared to the ABZ and MTF groups Table .Table 2There was a significant decrease in the mean diameter of hepatic granulomas in all treated groups compared to the matching positive control group. Compared to other treated groups, the combined group demonstrated significant decreases. The differences between each drug-treated group in the acute phase of the infection and its corresponding group in the chronic phase were not statistically significant Table .Multiple cellular granulomas composed of a collection of inflammatory cells, including macrophages, lymphocytes, and eosinophils, were observed in liver sections of the acute positive control group Fig. a. The chT. canis larvae in parenchymal tissue with few congested blood vessels. Nonetheless, these larvae did not induce any surrounding inflammatory cell infiltration  had significantly higher MMP-9 reactivity in brain tissues than the acute positive control group (G A/II). In addition, its expression in the positive control groups (G A/II and G C/II) Fig. a, b was G C/II group showed a more significant increment in levels of NO and MDA than the G A/II group as compared to the negative control (G I). All treated groups exhibited a significant decline in NO levels compared to the corresponding positive control group. However, the G C/V group showed a more substantial elevation in NO levels than all infected groups except G C/IV. MDA levels in all treated groups except G A/III were significantly lower than the matching positive control group, as depicted in Table G A/II and G C/II groups exhibited a more pronounced increase in the levels of SOD & catalase than the negative control Group (G I). Both SOD and catalase levels were significantly higher in the G A/IV and G A/V groups compared to the G A/III group. Similarly, both markers were significantly elevated in the G C/IV and G C/V groups relative to the G C/III group. In treated groups relative to G A/II and G C/II, the levels of SOD and catalase did not differ significantly between G C/IV and G C/V Table .P < 0.001), as shown in Table G A/II and G C/II had significantly lower levels of FGF21 compared to G I. All treated groups had significantly higher concentrations than the positive control groups. Additionally, levels of FGF21 in combined treated groups showed a significant increase in comparison with other treated groups. G A/II demonstrated a more significant increase in TNF-α levels than G C/II when compared to the negative control. However, G C/V demonstrated a higher decrease in TNF-α levels as compared to MTF- and ABZ-treated groups. TNF-α levels were decreased in G C/IV than in G A/IV. Moreover, G C/III had decreased levels of TNF-α than G A/III. There was a significant difference in TNF-α levels in all study groups . There was a statistically significant increase in its expression in the chronic positive control group compared to other groups (P < 0.001). However, the acute combined group showed a significant decrease compared to other studied groups. There were non-significant differences between acute ABZ and acute MTF (P < 0.420) as well as between chronic ABZ and chronic MTF (P < 1.0) (Fig. There were significant differences in relative liver TGF-β gene mRNA expression between all treatment groups and the corresponding positive control group (TF P < 0.20 as welCurrently, the efficacy of the available anti-toxocariasis drugs remains suboptimal. ABZ has emerged as one of the recommended drugs (Magnaval et al. T. canis larvae in the brains, livers, and muscles than administration of ABZ alone during the acute and chronic stages of the infection. Additionally, MTF-treated groups showed significant reductions compared to the positive control groups. To our knowledge, it is the first time to investigate the efficacy of MTF on toxocariasis. However, these results were not surprising as previous studies on the efficacy of MTF in the treatment of other parasitic infections reported its effectiveness in reducing parasite burdens. For instance, MTF could reduce the total number of larvae in the muscles of Trichinella spiralis-infected mice (Othman et al. Plasmodium yoelii-infected mice were significantly reduced by MTF administration (Miyakoda et al. In the current study, combined treatment was more effective on T. canis larval migration. Larvae actively moving through the liver generate tissue disorganization and granulomatous inflammation, which can result in granulomatous hepatitis (Kayes T. canis-infected mice with ABZ and MTF significantly decreased granuloma numbers and granuloma diameter and fibrosis compared to ABZ-treated groups. This observation was true for the infection’s acute and chronic phases. The decrease in granuloma counts paralleled the decrease in hepatic larval counts. Comparatively, the MTF-treated groups had significantly smaller granulomas than the positive control groups. Salama et al. (Schistosoma mansoni-infected mice.The liver is one of the visceral organs most frequently impacted in visceral is Kayes . In terma et al.  demonstrIn an effort to determine the underlying mechanisms by which MTF alleviates hepatic pathology, the redox status, TGF-β, and FGF21 levels in the hepatic tissues were measured. In comparison to the positive control groups, the MTF, and combined groups exhibited a significant reduction in oxidative stress, as evidenced by lower levels of MDA and NO and higher levels of SOD and catalase (Hou et al. T. canis-induced hepatic disease. Therefore, inhibiting NO production could aid in mitigating the damage caused by T. canis infection. This finding was demonstrated by Espinoza et al. (T. canis-infected rats.NO is a highly reactive chemical that plays a significant role in hepatic cell damage. Different cells, including hepatocytes, kupffer cells, macrophages, and endothelial cells, secrete it in the liver (Aksu et al. a et al. , who repFibrosis is considered to be primarily regulated by TGF-β signaling. According to Zhang  and Mi eIn contrast, FGF21 is predominantly expressed in hepatocytes, and its administration as a treatment has multiple direct and indirect effects on the liver (Coskun et al. T. canis-infected mice. TGF-β mRNA expression was enhanced in the infected untreated mice compared to the negative control. In acute and chronic phases of infection, Its expression was significantly decreased under the effect of combination therapy compared to the groups treated with either ABZ or MTF alone. These results are consistent with Li et al. (In the present study, we examined changes in the expression of TGF β-mRNA and FGF21 levels in the liver of i et al.  and Lu ei et al. , who illRegarding levels of FGF21 in the current study, all treatment groups showed significant increases compared to the positive control groups. MTF and combined treated groups showed the highest levels. As previously reported by Nygaard et al. , metformT. canis infection, the larvae travel across all body organs. As the infection progresses, they tend to leave the different organs, such as the liver and lungs, and frequently build up in the muscles and the brain (Chou et al. During the course of T. canis infection has shown that the absence of inflammation around the larvae in the brain is a common observation in the mouse model (Liao et al. Previous research on T. canis resulted in a significant increase in the levels of TNF-α inside the brain tissues of the infected mic. Similarly, Othman et al. (T. canis-infected mice with the progression of the infection. TNF-α is a pro-inflammatory cytokine that is released by a wide variety of brain cells such as neurons, astrocytes, microglia, and oligodendrocytes. Its receptors are distributed throughout the CNS (Probert The present investigation revealed that infection with n et al.  showed u Probert . Its exp Probert .Although increased production of pro-inflammatory cytokines, such as TNF-α, is helpful in boosting clearance of invasive infections and phagocytosis of cell debris, prolonged inflammatory processes can cause harmful brain damage and neurodegeneration (Lucas et al. A comprehensive investigation was conducted to examine the function of MMP-9 in the etiology of various neurological diseases. For instance, previous studies have shown its involvement in several types of brain traumas, including traumatic, hemorrhagic, and ischemic injuries (Chaturvedi and Kaczmarek In the present research, an investigation was conducted to compare the expression of MMP-9 in the brain of mice across various study groups. The results revealed a substantial increase in MMP-9 expression in the positive control group compared to the negative control group. A decrease in expression was seen in mice subjected to treatment with either MTF or a combination of treatments, regardless of whether it was during the acute or chronic stages of the infection. The findings of this study demonstrate the significant impact of MTF on the expression of MMP-9. Prior research conducted by Zhang et al.  demonstrToxocara canis larvae induces dynamic changes in the systemic immune response of the host. In the current study, T. canis infection induced a predominant decrease in IL-12 and IFN-γ levels and an increase in IL-4 along the course of the infection. Additionally, IL-10 levels increased significantly more during the chronic phase than during the acute phase. These findings are supported by prior research (Kuroda et al. Migration of T. canis larvae use crucial immunological mechanisms, including the suppression of IL-12 and IFN-γ, as survival mechanisms. This suppression hinders the recruitment of macrophages and prolongs the lifespan of the parasite (Kuroda et al. T. canis from inhibiting IL-12 and contributes to the body’s anti-infection response. This result is consistent with previous research indicating that an increase in IL-12 and IFN-γ may enhance the protective host response against T. canis (Pilarczyk et al. T. canis infection in mice. It decreased recovery in the liver, brain, and muscles of developing larvae. Combined therapy was superior to ABZ alone in treating hepatic and cerebral pathologies. In addition, MTF was able to regulate the host’s immune response and oppose the immunomodulatory processes induced by T. canis infection. Identical effects were observed during both the acute and chronic phases of infection. We hypothesize that metformin exerts its anti-toxocara effects by modulating the immune response and ameliorating infection-related pathology. Therefore, MTF should be considered an adjuvant therapy for the treatment of experimental toxocariasis.Our findings indicate that MTF has a positive effect on reducing the pathological changes caused by


In the surveillance group, high levels of dd-cfDNA (>0.5%) from second month post-transplantation were correlated with non-increasing eGFR 1 year post-transplantation. The study used AlloSeq kit for kidney transplant surveillance for first time and confirmed dd-cfDNA’s ability to detect rejection and monitor treatment, as well as to predict worse long-term outcomes regarding eGFR.In our prospective, unicenter cohort study, we collected blood samples from 30 newly kidney transplanted patients, at month 1, 2, 3, and 5 for dd-cfDNA analysis, along with creatinine/eGFR and DSA monitoring, and from 32 patients who underwent an indication biopsy and whose dd-cfDNA levels were measured at the time of biopsy and 1 month afterwards. Fourteen of 32 (43.8%) patients in the biopsy group were diagnosed with TCMR and 5 of 32 (15.6%) with ABMR. Dd-cfDNA proved to be better than creatinine in diagnosing rejection from non-rejection in patients who were biopsied. When a dd-cfDNA threshold of 0.5% was chosen, sensitivity was 73.7% and specificity was 92.3% . In rejection patients, levels of dd-cfDNA prior to biopsy  decreased substantially after initiation of treatment with median returning to baseline already at 1 month (0.33%, 0.21–0.51,   Dd-cfDNA and eGFR for each month was assessed. There were two categories of patients: those with a high dd-cfDNA (any measurement above 0.5%) and those with a low dd-cfDNA . A two-way repeated-measures analysis of variance (ANOVA) was performed for the analysis.The demographics of the 62 patients enrolled in our study depict a population of high immunological risk . An ABO p = 0.3 . The Youden’s index for dd-cfDNA was 0.58%. When a dd-cfDNA threshold of 0.5% was chosen, sensitivity was 73.7% and specificity was 92.3%.Using 32 for cause biopsies from 32 patients with biopsy-paired dd-cfDNA results, the association between dd-cfDNA levels and any allograft rejection status was evaluated. Even though changes in serum creatinine make up the largest proportion of reasons for a biopsy in our study, there was no statistically significant difference in the median creatinine in patients with a no rejection biopsy  and patients with Banff-defined rejection ; p = 0.3 . The AURp < 0.001. TCMR was diagnosed in 14 biopsies. Patients with TCMR, compared to nonrejection patients, had a median dd-cfDNA value of 0.52%; p = 0.038.ABMR was diagnosed in 5 biopsies. Among these patients, compared to non-rejection patients, the median dd-cfDNA was 13%; p < 0.001) and more than 12 times higher odds of TCMR compared to those with dd-cfDNA lower than 0.5% (p = 0.031)  , 4.p = 0.0036. The difference was even more significant when comparing median dd-cfDNA levels at month 2  to median levels at month 0 (p = 0.0007).Dd-cfDNA kinetics were evaluated in 19 recipients diagnosed with rejection . In ordeAccording to our study, the median value of dd-cfDNA for 30 surveillance patients from the first 5 months post-transplantation was 0.23% (IQR: 0.18%–0.36%). Moreover, nine transplant recipients who were initially enrolled in the surveillance group had median dd-cfDNA of 0.33% (IQR: 0.24%–0.37%) before being referred for a graft biopsy and being ‘transferred’ to the biopsy group. These findings suggest that median dd-cfDNA levels returned to baseline levels already at the first month after anti-rejection treatment, while dd-cfDNA levels at month 2 were similar to the median dd-cfDNA levels of the surveillance group.p = 0.004) compared to recipients with at least one high measurement of dd-cfDNA (≥0.5%) (p = 0.725), whose eGFR did not seem to rise that efficiently 1 year post-transplantation. However, the mean value of eGFR has not been significantly different between the two groups in month 12.In the surveillance group of the 30 newly transplanted recipients, an effort was made to assess how the elevation of dd-cfDNA affects changes in eGFR 1 year post-transplantation. The two-way repeated-measures ANOVA was run on the eGFR at month 5 and 12 in two groups of 22  and 8 (dd-cfDNA ≥ 0.5% at least in one measurement—month 1 excluded) newly transplanted patients. Groups were defined according to the percentage of dd-cfDNA (cut-off point = 0.5%) and the number of measurements . A diffeAs mentioned above, nine of the 32 renal recipients who underwent a biopsy had been enrolled in the surveillance group at the beginning of the study but were shifted to the biopsy group after an indication for a for cause biopsy was received. All these recipients were biopsied after the second month post-transplantation. As a result, nine patients had at the end of the study at least two monthly dd-cfDNA measurements prior to the biopsy event. We decided to compare the first two measurements of these recipients to the first two dd-cfDNA measurements of the 30 surveillance patients who managed to complete 5 months post-transplantation without the need of a for cause biopsy.p = 0.001) compared to those who needed one the first months post-transplantation  and dd-cfDNA (%), and similar results were obtained for composite diagnosis of ABMR . In a GeIt has been reported in the recent ADMIRAL study that patients with clinical ABMR had significantly higher levels of dd-cfDNA (2.2% versus 0.34%) , whereasGiven the small number of participants, we decided to include the 2 patients diagnosed with borderline rejection in the TCMR group , keeping in mind although the heterogeneous injury within this diagnosis, as Stites et al.  proved bIt was calculated that our optimal threshold for dd-cfDNA using AlloSeq cfDNA assay in order to discriminate rejection from non-rejection was 0.58%, which complies with the ADMIRAL study, whose Youden Index for dd-cfDNA was 0.69%, while the AUROCs in the two studies when using the same threshold were similar .Former sThe delta between serial dd-cfDNA was also associated with increased possibility for an indication biopsy, suggesting that dd-cfDNA alterations can be an alarming sign for the allograft quiescence. Using the dd-cfDNA as an indicator, Anand et al. showed thp = 0.0036.Wolf-Doty et al.  have monp = 0.004), while those with at least one high dd-cfDNA value excluding first month did not show the same increase. It should be noted, however, that due to the short follow-up period, the mean value of eGFR has not been significantly different between the two groups after month 12. Bu et al. [It was concluded that recipients with low dd-cfDNA levels showed a clear increase in eGFR after month 12 (u et al.  demonstru et al.  stated tIt has been demonstrated by Aubert et al.  that dnDPreviously mentioned, the major limitation of our study was the small sample size, which prevented the correlation of dd-cfDNA with DSA formation, and in combination with some very high values in ABMR patients, led to large confidence intervals and high diagnostic odds ratios regarding rejection. Nevertheless, these limitations did not affect the AUROC performance or usefulness of the biomarker as a monitoring tool. A longer follow up period could also strengthen the correlation between high dd-cfDNA and worse outcome regarding eGFR over time. Moreover, in view of the evident multifactoral value of considering dd-cfDNA as part of the clinical assessment of the patient, further research is required to determine the optimal monitoring interval. The novelty of this study is that Alloseq dd-cfDNA kit was used locally for dd-cfDNA testing and useful clinical data was provided about how this kit performed in the real-world.In summary, this report using AlloSeq cfDNA kit for local testing confirms large multicenter service-based trials regarding dd-cfDNA’s validity as a tool to surveil the allograft quiescence, to detect rejection and monitor treatment, as well as to predict outcomes regarding graft survival. However, since dd-cfDNA is positioned to be added within the existing panel of current routine testing rather than be used as single information for taking clinical decision, it is undisputed that further research combining dd-cfDNA with other biomarkers is required to improve our diagnostic tools in relation to allograft rejection.